It looks that some of my cell lines show two bands for vinculin on WB. The two bands are very close to each other, and they are on the right MW.
I washed the cells with PBS for several times, so it shouldn't be caused by FBS.
Hello dear Wjkp
It might be because of several possible reasons mentioned below:
It might be possible that your target protein has been cleaved or digested. In order to stop it, you can try using freshly prepared protease inhibitors.
It might be another protein bearing the same/similar epitope has been detected by your antibody. Solution: try with the antibody of a different company.
It can be because of some post-translation modification of your protein.
Or else, you might be using too high concentration of primary antibody, as a result, non-specific band.
I hope the clue will help you to understand your problem.
Vinculin has a near relative, a spliceosome variant known as meta-vinculin. Did you check for that, sir? I am not an expert in the field but check for it. It is said that meta-vinculin could be longer in nature, leading to lesser mobility and even probably showing a lighter band when compared to that of the lower band (depending on the marker you use, of course). Viculin is said to have a mol. wt. of 120 and meta-vinculin about 150. So, please do let me know if this was in any way helpful.
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